F4/80 Antibody Staining Protocol for Immunohistochemistry


Description: MCAP497 recognises the mouse F4/80 antigen, a 160kD glycoprotein expressed by murine macrophages. The antibody binds to macrophages from different sites including the peritoneal cavity, lung, spleen and thymus, to blood monocytes and to macrophages derived from bone marrow precursors in culture. The antigen is not expressed by lymphocytes or polymorphonuclear cells.


Primary Antibody

Name: F4/80 Antibody

Clone: CI:A3-1, Rat anti-Mouse

Supplier: AbD Serotec

Catalog Number: MCAP497

Dilution: 1:50 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 minutes/room temperature

Antigen Retrieval
Device: Incubator
Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101)
Heat/Cool Temperature: 37 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods
Standard Method: ABC Method or LSAB Method
Enhanced Method: Polymeric Methods

Chromogen Substrate
Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Membrane/cytoplasmic
Images: Search image

Additional Information:
Tissue Type: Spleen, lung, thymus.
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Spleen, lung, thymus.
Negative Control: Omit primary antibody, isotype control or absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary


No need to perform antigen retrieval on frozen sections